The molecular mechanisms involved in the regulation of the human intestinal NaCI absorption, mediated by the concerted action of dual ion exchanges of Na/H+ and Cl-/HCO3- are not well understood. Our prior studies of the human small intestine and colon suggest that the Na+/H+ exchanger, NHE3, may be the predominant isoform involved in Na+ absorption in the small intestine, whereas NHE2 is important for colonic Na+ absorption. It is, therefore, crucial to investigate the regulation of human Na+/H+ exchangers for a better understanding of normal human intestinal physiology and to understand the molecular basis of diarrheal disorders. Our studies of the NHE3 promoter showed that NHE3 expression is regulated by a number of transcription factors including AP-2, Egr-1 and Sp1 family members. The regulation of NHE3 exchanger activity by different stimuli such as serotonin, nitric oxide, IFN-y, PMA and EPEC infection was also demonstrated. We hypothesize that cis-elements in the NHE3 promoter region and their cognate transcription factors mediate the processes involved in NHE3 regulation during basal transcription, tissue-specific expression and intestinal inflammation and propose the following three specific aims to address them. Specific Aim 1 will elucidate the role of inflammatory mediators in transcriptional regulation of NHE3 by investigating the mechanisms involved in the regulation of the NHE3 gene expression by EPEC infection and associated inflammatory mediators such as TNFalpha and IFN-y and by characterizing the cis-regulatory elements and their corresponding trans-acting factors that mediate the effects of these agents. Our Specific Aim 1 will interact with Project 1 and Project 4. Specific Aim 2 is designed to determine the molecular mechanisms involved in the transcriptional regulation of the NHE3 gene in response to mitogenic stimulus PMA, through characterization of the putative PMA- response elements and transcription factors Egr-1, Sp1 and Sp3. Specific Aim 3 will identify the cis- and trans-acting factors that regulate cell-specific expression of the NHE3 gene through epigenomic mechanisms utilizing, C2/bbe, Caco-2, NCM460, LnCap, PCS, HepG-2, MDCK cells as well as NIH3T3 mouse fibroblast cell line.